review open access pharmacokinetic and pharmacodynamic

Karahoca and Momparler Clinical Epigenetics 2013, 5:3http://www.clinicalepigeneticsjournal.com/content/5/1/3

REVIEW Open Access

Pharmacokinetic and pharmacodynamic analysisof 5-aza-2’-deoxycytidine (decitabine) in thedesign of its dose-schedule for cancer therapyMetin Karahoca1 and Richard L Momparler1,2*

Abstract

5-Aza-2′-deoxycytidine (5-AZA-CdR, decitabine), an epigenetic drug that inhibits DNA methylation, is currently usedto treat myelodysplastic syndrome (MDS), and is under investigation for treating acute myeloid leukemia (AML) andother malignancies. 5-AZA-CdR can reactivate tumor suppressor genes silenced by aberrant DNA methylation, afrequent event in all types of cancer. Because this epigenetic change is reversible, it is a good target for 5-AZA-CdRtherapy. We have reviewed the preclinical data of 5-AZA-CdR to analyze the concentrations and exposure timesrequired to eradicate cancer stem cells. We analyzed the dose-schedules used in animal models that show potentantineoplastic activity of 5-AZA-CdR. We attempted to correlate the preclinical data with the responses obtained inclinical trials of 5-AZA-CdR in patients with cancer. The pharmacokinetics and drug distribution of 5-AZA-CdR arekey parameters because adequate therapeutic drug levels are required to eliminate cancer stem cells in allanatomic compartments. The plasma half-life of 5-AZA-CdR in humans is approximately 20 minutes due to the highlevels in the liver of cytidine deaminase, the enzyme that inactivates this analogue. This provides a rationale to usean inhibitor of cytidine deaminase in combination with 5-AZA-CdR. Low-dose 5-AZA-CdR is effective for MDS andAML and can induce complete remissions (CR). However, maintenance of CR with low-dose 5-AZA-CdR is difficult.Based on analyses of preclinical and clinical data, low dose 5-AZA-CdR has the potential to be an effective form oftherapy in some patients with cancer. For patients who do not respond to low dose therapy we recommenddose-intensive treatment with 5-AZA-CdR. Patients who are candidates for intensive dose 5-AZA-CdR should have agood bone marrow status so as to permit adequate recovery from myelosuppression, the major toxicity of 5-AZA-CdR.Solid tumors are also interesting targets for therapy with 5-AZA-CdR. Both low dose and intensive therapy with5-AZA-CdR can reduce the proliferative potential of tumor stem cells in animal models. We propose novel doseschedules of 5-AZA-CdR for investigation in patients with cancer. The full chemotherapeutic potential of 5-AZA-CdR totreat cancer merits further clinical investigation and can only be realized when its optimal dose-schedule is determined.

Keywords: Decitabine, 5-aza-2’-deoxycytidine, Epigenetics, AML, MDS, DNA methylation, Pharmacokinetics,Pharmacodynamics

Introduction5-Aza-2′-deoxycytidine (5-AZA-CdR, decitabine) wasfirst synthesized in 1964 by Pliml and Sorm [1] and itspotential activity in leukemia was reported in 1968 bySorm and Vesely [2]. 5-AZA-CdR is an analog of thenatural nucleoside 2′-deoxycytidine in which the carbon

* Correspondence: [email protected]épartement de Pharmacologie, Université de Montréal, Montréal, Québec,Canada2Service of Hematology and Oncology, Centre de recherche,CHU-Saint-Justine, 3175 Côte Sainte-Catherine, Montréal, Québec H3T 1C5,Canada

© 2013 Karahoca and Momparler; licensee Biothe Creative Commons Attribution License (htdistribution, and reproduction in any medium

in the 5-position of the cytosine is replaced by nitrogen.Preclinical studies in rodents indicated that 5-AZA-CdRis a more potent antileukemic agent than cytosine arabi-noside (ARA-C) [3,4]. In 1979, Taylor and Jones [5]reported that 5-AZA-CdR could induce cells in cultureto differentiate into different phenotypes and this activitycorrelated with its inhibition of DNA methylation. Thefirst review on the pharmacologic properties of 5-AZA-CdR was published in 1979 [6] and the first clinical trialon 5-AZA-CdR in patients with acute leukemia waspublished in 1981 [7].

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Figure 1 Intracellular metabolism of 5-AZA-CdR. 5-AZA-CdR istransported into the cell by the equilibrative-nucleoside transportsystem. 5-AZA-CdR converts into its triphosphate form byphosphorylation and binds covalently to the DNA, where itblocks DNMTs and causes demethylation of DNA. 5-AZA-CdR,5-aza-2′-deoxycytidine; DNMTs, DNA methyltransferases.

Karahoca and Momparler Clinical Epigenetics 2013, 5:3 of 16http://www.clinicalepigeneticsjournal.com/content/5/1/3

Although the unique demethylating capacity of 5-AZA-CdR has been known for many years, its approval for thetreatment of cancer took a long time, perhaps due to alack of understanding of the importance of epigeneticchanges in malignancy during the early years of develop-ment [8]. 5-AZA-CdR was approved for the treatment ofmyelodysplastic syndrome (MDS) in 2006 and shows anti-leukemic activity against acute myeloid leukemia (AML)as well [8,9]. Its clinical activity against solid tumors isunder investigation. 5-AZA-CdR also displays some effect-iveness in the treatment of sickle cell disease, which isnonmalignant [10]. Low-dose 5-AZA-CdR was proposedto be effective for treating MDS due to its epigeneticaction, whereas higher doses were too toxic due the poorhematologic status of these patients [11]. The objectives ofthis review is to analyze preclinical and clinical data on5-AZA-CdR and develop a rationale for optimization ofits dose schedule for the treatment of cancer.

ReviewMode of action and cellular metabolismRecent progress on elucidating the important role of epi-genetics in the development of malignancy has generatedmore interest in the chemotherapeutic potential of 5-AZA-CdR for the treatment of cancer [12,13]. ‘Epigenetics’ refersto the study of heritable changes in gene expression thatoccur without changes in the DNA sequences [14]. Epi-genetic regulation of gene expression is modulated bychanges in DNA methylation, covalent histone modifica-tions, and microRNA [15-17]. Hypermethylated tumorsuppressor genes represent one of the most consistenthallmarks of human cancers [12,18]. Loss of the expres-sion of tumor suppressor genes can lead to a loss of regu-lation of cancer cell growth [19-21].DNA methylation usually occurs at the 5-position of

the cytosine ring within cytosine-phosphate-guanine(CpG) dinucleotide by a transfer of the methyl groupfrom S-adenosyl-L-methionine [22]. DNA methyltrans-ferases (DNMTs) catalyze this reaction. The methyla-tion pattern from the parental DNA is copied onto thenewly synthesized DNA strand by the maintenancemethyltransferase DNMT 1. In embryonic stem cells andtumor cells, methylation of previously unmethylated DNA(de novo methylation) is catalyzed by DNMT 3a or DNMT3b [23]. To inactivate transcription, methylation usuallyoccurs in the CpG islands in the promoter-exon regions oftarget genes. Half of all genes harbor CpG islands in theirpromoters [24]. In human DNA, approximately 50% to70% of CpG dinucleotides are methylated [25,26]. Duringnormal embryonic development, cytosine methylation isessential for establishing tissue-specific gene expression, si-lencing imprinted genes, and inactivating the X chromo-some. Methylation also protects against the transcriptionof parasitic elements [27].

Both 5-AZA-CdR and 5-azacytidine have been reportedto inhibit the expression of the de novo DNA methylatingenzymes, DNMT3B [28,29]. DNMT3A-DNMT3B doublenull embryonic stem cells are highly resistant to 5-AZA-CdR as compared to single null or wild type cells [30].Mutations in DNMT3A have been identified in AML andMDS [31,32]. These DNMT3A mutations are associatedwith a poor outcome for both AML and MDS patients. Arecent report showed that DNMT3A plays a role in silen-cing self-renewal genes in hematopoietic stem cells so as topermit hematopoietic differentiation [33]. Preliminary dataindicate that AML patients with low DNMT3A activitymay benefit from treatment with 5-AZA-CdR [34]. The fullrole of DNMT3A and DNMT3B in leukemogenesis stillremains to be clarified.5-AZA-CdR is a prodrug that must be activated by phos-

phorylation. The metabolism of this analog is summarizedin Figure 1. Due to the function of the nucleoside transportsystem, 5-AZA-CdR rapidly reaches its equilibration statebetween the extracellular and intracellular compartments,as indicated by the short alpha half-life of five minutes [35].The cellular uptake of the drug is realized by an equili-

brative nucleoside-specific transport mechanism [36-39],which is followed by phosphorylation and incorporationof the drug into DNA, resulting in potent inhibition ofDNMT.


Since 5-AZA-CdR is a prodrug it must be activated bydeoxycytidine kinase to its monophosphate form and byother kinases to its triphosphate form, which is then incor-porated into DNA by DNA polymerase [5,40-43]. The in-hibition of DNA methylation is due to formation of acovalent complex between 5-AZA-CdR-DNA and DNMT1at CpG methylation sites, resulting in the inactivation ofthis enzyme [44,45]. DNMT1 inhibition results in DNAhypomethylation, gene activation and the induction of cel-lular differentiation, senescence, and/or apoptosis [40-42].5-AZA-CdR does not block the progression of G1-

phase cells into S-phase [46]. Flow cytometry investiga-tions revealed that 5-AZA-CdR slows the progression ofcells into S-phase, but it does not block cell progressionthrough their cycle [47], unlike the related nucleosideanalogs 5-azacytidine and ARA-C [48]. One of the keybiochemical markers of 5-AZA-CdR is the p15 tumorsuppressor gene, which is frequently hypermethylated inMDS and AML patients and can be reactivated by thisanalog [49]. Table 1 summarizes key genes that aresilenced by DNA methylation in different types of canceras published in the review by Esteller [15].

In vitro antineoplastic actionLeukemiaIn vitro studies of 5-AZA-CdR demonstrate that itinduces differentiation, apoptosis, and senescence inleukemic cells [40-42,50-52]. One proposed mechanism ofthis action is that 5-AZA-CdR exposure reduces the ex-pression of c-myc, an oncogene that prevents the terminaldifferentiation of cells [53]. 5-AZA-CdR also maintainshematopoietic precursor self-renewal [54]. Leukemic cellscan undergo phenotypic modification after exposure to5-AZA-CdR, as indicated by the increased expression ofseveral antigen markers [55,56]. 5-AZA-CdR enhances thegraft-versus leukemia effect, which has the potential to

Table 1 Silencing by DNA methylation of key genes indifferent types of cancera

Genes Type of cancer

p16, p14, RARβ2, SFRP1 Colon cancer

p16, p14 Esophageal cancer

p14, hMLH1 Stomach cancer

SOCS1, GSTP1 Liver cancer

ER, BRCA1, E-cadherin, TMS1 Breast cancer

BRCA1 Ovarian cancer

GSTP1 Prostate cancer

p16, DAPK, RASSFIA Lung cancer

p16, TPEF/HPP1, miR-127 Bladder cancer

VHL Kidney cancer

p15, EST1, ID4 Leukemia

p16, p73, MGMT Lymphomaamodified from Esteller M. [15]. p16, p16INK4a; p14, p14ARF; p15, p15INK4b.

increase the immunologic therapeutic efficacy of allogen-eic transplantation [57]. 5-AZA-CdR also induces theupregulation of DNA repair genes and angiogenesis in-hibitor genes [58]. These changes can suppress the devel-opment of malignancy. In vitro studies of 5-AZA-CdR alsoillustrate its potent antileukemic activity [59,60]. At equi-molar concentrations, 5-AZA-CdR more potently reducescolony formation than either 5-azacytidine or ARA-C [61].5-AZA-CdR also has greater DNA-hypomethylating activ-ity than 5-azacytidine [61]. In vitro studies on the inductionof the loss of clonogenicity by 5-AZA-CdR on humanleukemic cell lines of different phenotypes are summarizedin Table 2. The 50% inhibitory concentration (IC50) of col-ony formation for a one-hour exposure to 5-AZA-CdRwas approximately 10 μM for HL-60 (myeloid), Molt-3(T-cell) and RPMI-8392 (B-cell) leukemic cell lines. For a24-hour exposure, the IC50 of 5-AZA-CdR is in the rangeof 0.1 μM for these leukemic cell lines. This observationillustrates the importance of the duration of treatment withrespect to the antileukemic activity of 5-AZA-CdR.

In solid tumorsSeveral studies on different type of tumors demonstratedan association between DNA hypermethylation andDNMT overexpression [62-64]. The precise molecularmechanism of aberrant DNA methylation, however,remains to be clarified. The first tumor suppressor genefound to undergo silencing as a result of promotermethylation was the Rb gene in retinoblastoma tumors,followed by numerous other tumor suppressor genes[65-67]. 5-AZA-CdR reactivates many genes that sup-press malignancy and are silenced by aberrant DNAmethylation in solid tumors (Table 1). 5-AZA-CdR reac-tivates the tumor suppressor gene VHL in human renalcarcinoma cell lines and the expression of the p16/CDKN2 tumor suppressor gene, which prevents theentry of tumor cells into the S-phase of the cell cycle, indifferent tumor cell lines [20,68-72]. The mRNA expres-sion of the p53 gene increases in hepatoma cell lines after

Table 2 In vitro concentrations of 5-AZA-CdR thateradicate the proliferative potential of leukemic cells

Leukemic cell lines IC50 Duration ofexposure

References

HL 60 (myeloid) 100 μM 1 hour [59]

Molt-3 (T-cell) 10 μM 1 hour [59]

RPMI 8392 (B-cell) 10 μM 1 hour [59]

HL60 leukemic cells 0.05 to 0.1 μM 24 hours [60]

L1210 leukemic cells 0.075 μM 18 hours [60]

Molt-3 (T-cell) about 0.1 μM 24 hours [59]

HL 60 (myeloid) about 0.1 μM 24 hours [59]

RPMI 8392 (B-cell) about 0.1 μM 24 hours [59]

1 μM = 228 ng/ml. 5-AZA-CdR, 5-Aza-2′-deoxycytidine; IC50, half maximalinhibitory concentration.


treatment with 5-AZA-CdR [73]. Methylation of hMLH1,a mismatch repair gene that confers chemoresistance tocertain anticancer agents, is reactivated by 5-AZA-CdR inovarian and colon cancer cell lines [74,75]. 5-AZA-CdRreactivates CDKN2a (p16INK4a) in lung cancer, BRCA1in breast cancer, and MGMT in glioblastomas [76-79].Expression of unmethylated genes, such as Apaf-1 andCDKN2D in malignant melanoma and lung cancer, isenhanced after 5-AZA-CdR treatment, indicating thatgene activation can occur by indirect mechanisms [80,81].5-AZA-CdR reduces natural killer cell responsiveness,which may be used in therapeutic strategies to target ma-lignant cells by an immune mechanism [82].Preclinical studies using colony assays indicate that

5-AZA-CdR is an active antineoplastic agent againstmany different tumor cell lines [60,83-88]. These stud-ies demonstrated that the demethylation of genesinvolved in cell cycle control inhibits the growth ofvarious tumors [66,79,89]. The in vitro studies on theantineoplastic activity of 5-AZA-CdR in tumor celllines are summarized in Table 3. For example, theIC50 is approximately 4 μM for a two-hour exposurein fibrosarcoma cells and approximately 0.5 μM for afour-hour exposure in Calu-6 lung carcinoma cells[60,88]. For a 48-hour exposure, the IC50 is in therange of 0.1 μM for different tumor cell lines (Table 3).As observed with leukemic cells, the duration of treat-ment is a key factor with respect to the antineoplasticactivity of 5-AZA-CdR.

In vivo antineoplastic actionThe antineoplastic activity of 5-AZA-CdR was firstdemonstrated in mouse models of acute leukemia [2]. In

Table 3 In-vitro concentrations of 5-AZA-CdR thateradicate proliferative potential of solid tumor cells

Tumor cell lines IC 50 Duration oftreatment

References

HS-SY-II synovial sarcoma 1.3 μM 96 hours [84]

SYO-1 synovial sarcoma 0.9 μM 96 hours [84]

KB oropharyngeal cancer 0.5 μM 96 hours [85]

A549 lung adenocancer 0.49 μM 96 hours [85]

LoVo colon cancer 0.4 μM 96 hours [85]

LoVo-DX colon cancer 0.1 μM 96 hours [85]

MDA-MB- 435 breast cancer 0.2 μM 48 hours [83]

Hs578T breast cancer 0.13 μM 48 hours [87]

MCF-7 breast cancer 0.13 μM 48 hours [87]

MDA-MB- 231 breast cancer 0.013 μM 48 hours [83]

EMT6 mammary cancer 0.22 μM 18 hours [60]

Calu-6 lung cancer 0.44 μM 4-8 hours [60]

A(T1)C1-3 fibrosarcoma 4.38 μM 2 hours [88]

1 μM= 228 ng/ml. 5-AZA-CdR, 5-Aza-2′-deoxycytidine; IC50, half maximalinhibitory concentration.

mice with murine L1210 leukemia, the activity of 5-AZA-CDR markedly increases with an increased dose and ex-posure time [60,61]. In a preclinical study using the L1210mouse leukemia model, the curative dose of 5-AZA-CdRadministered as a 15-hour infusion was 20 mg/kg. Theantileukemic activity of 5-AZA-CdR, 5-azacytidine, andARA-C was compared in the L1210 leukemia mousemodel [61]. The mice were administered these nucleosideanalogs in a 15-hour continuous intravenous (i.v.) infusionto obtain an effective plasma concentration of the drugthat persisted longer than the cell cycle of these leukemiacells (12 hours). At equitoxic doses, 5-azacytidine (11.7mg/kg) increased the life span of the mice by 63%,whereas 5-AZA-CdR (10.1 mg/kg) increased the life spanof the mice by 384%. At higher doses, 5-AZA-CdR curedthe mice of leukemia, but 5-azacytidine or ARA-C didnot. Because the cell cycle of L1210 leukemic cells isaround 12 hours in duration, the 15-hour infusion of theseanalogs was predicted to produce 100% cell kill in thismouse model because all of the leukemic cells enteredS-phase during the treatment. Analysis of the survivingleukemic cells revealed that they were not resistant toARA-C.In a rat model of myeloid leukemia, 5-AZA-CdR

increased survival in a dose-related manner [4]. In thismodel, the antileukemic action of 5-AZA-CdR exceededthat of ARA-C. The hematologic toxicity of 5-AZA-CdRwas evaluated in this rat model by the colony forming unitin the spleen (CFU-S) assay [4]. The maximum number ofhematopoietic stem cells killed at high doses of 5-AZA-CdR was the same as that at low doses, indicating that theresponse reaches a plateau upon dose escalation. This issolid evidence that normal resting hematopoietic stemcells survive high-dose treatment with 5-AZA-CdR. Thedifference between hematopoietic stem cell toxicityand antileukemic effects favors treatment with highdoses of 5-AZA-CdR because the dose that causesmaximal leukemic cell kill is not more toxic to restinghematopoietic stem cells.5-AZA-CdR displays significant antineoplastic activity

against tumors in a mouse model. In mice bearing EMT6mammary tumors, 5-AZA-CdR at a dose of 15 mg/kgproduces a 2-log reduction in clonogenic survival asmeasured by excision of the tumor, preparation of asingle-cell suspension and analysis of survival by in vitrocolony formation [60]. A higher dose of 30 mg/kg pro-duces a 3-log reduction in clonogenic survival. Theseobservations illustrate the effectiveness of intensive dosesof 5-AZA-CdR for tumor therapy.

Pharmacokinetics of 5-AZA-CdRMethods used to determine concentrations of 5-Aza-CdRSeveral methods are used to determine 5-AZA-CdRconcentrations, including bioassays, HPLC, and HPLC


combined with mass spectrometry (HPLC/MS). Amongthese methods, HPLC/MS is the most sensitive followedby bioassays; both of these methods are superior to HPLCalone for detecting low concentrations of 5-AZA-CdR.The lower limit of detection of 5-AZA-CdR is in the rangeof 0.01 μM. For pharmacokinetic studies, HPLC/MS is themethod of choice for detecting the plasma concentrationof 5-AZA-CdR followed by the bioassay and HPLC meth-ods (Table 4) [7,36,90-92].

Pharmacokinetic dataThe moderate chemical instability of 5-AZA-CdR is im-portant and dependent on both temperature and pH[93]. Therefore, this agent must be carefully formulated.The instability is due to the opening of the 5-azacytosinering between positions 5 and 6 to form N-(formylamidino)-N′-β-D-2-deoxyribofuranosylurea (NFDU). This reaction ishighly reversible in favor of 5-AZA-CdR and results in aminimal loss of pharmacologic activity. When NFDUdecomposes irreversibly via loss of the formyl moiety toform N′-β-D-2-deoxyribofuranosyl-3-guanylurea (DGU),there is a complete loss of pharmacologic activity. This re-action occurs rapidly in alkaline solutions and increaseswith elevation of the temperature. At 24°C, the 50% de-composition rates (D50s) of 5-AZA-CdR to DGU at pH7.0 and 8.5 are 22 hours and 5 hours, respectively. At37°C, the D50s at pH 7.0 and 8.5 are 12hours and 2 hours,respectively. At 5°C and pH 7.0 to 7.4, the decompositionof 5-AZA-CdR to DGU is minimal (<1%). To formulate5-AZA-CdR, we recommend using 5 to10 mM potassiumphosphate, pH 7.0 to 7.4 and storage at 5°C until clinicaluse. A fresh solution of 5-AZA-CdR can be preparedevery eight hours. During the infusion of 5-AZA-CdR topatients, we recommend using an ice pack to maintain thetemperature at 10°C [93]. After i.v. administration of5-AZA-CdR, its binding to plasma protein is less than 1%.There is a rapid distribution of 5-AZA-CdR between theextra- and intracellular compartments after i.v. injection,as indicated by its alpha half-life of five minutes [35].5-AZA-CdR penetrates the blood–brain barrier. The level

of 5-AZA-CdR in the cerebrospinal fluid (CSF) reaches ashigh as 50% of its plasma level during a continuous infusion

Table 4 Comparison of methods to determineconcentration of 5-AZA-CdR

Method Limit sensitivity for detection References

Bioassay 5 nM [7]

6 nM [36]

HPLC 750 nM [90]

HPLC/MS 10 nM [91]

5 nM [92]

1 μM = 228 ng/ml. 5-AZA-CdR, 5-Aza-2′-deoxycytidine; HPLC, highperformance liquid chromatography; HPLC/MS, Hjgh performance liquidchromatography/mass spectrometry.

[35]. The beta plasma half-life of 5-AZA-CdR in adults isapproximately 15 to 25 minutes due to very high levels ofcytidine deaminase in the human liver and spleen [94,95]In pediatric populations, the beta half-life is 10 to 15 min-utes, which is shorter than that of adults, probably due tothe higher cytidine deaminase activity in the liver andspleen of children [7,90]. Repeated administration of5-AZA-CdR in patients with MDS as a 3-hour infusion of15 mg/m2 every eight hours for three days does not resultin systemic accumulation of the drug, and the pharmaco-kinetic parameters remain unchanged from cycle to cycle[92]. An illustration of the plasma pharmacokinetics ofthe dose schedule that is currently used to treat MDS andAML is shown in Figure 2. 5-AZA-CdR is cleared fromthe systemic circulation rapidly. 5-AZA-CdR is inactivatedprimarily through deamination by cytidine deaminasein the human liver and spleen [94,95]. The half-life of5-AZA-CdR is related to the blood flow into the liver. Itsclearance exceeds the total renal capacity, which suggestsan important role of non-renal elimination. The clearancerate has been reported as approximately 125 ± 20 ml/min/kg [36]. The urinary excretion of unchanged 5-AZA-CdRis low, typically <1% of the total dose given to the patients[36]. Oral formulations of 5-AZA-CdR are currentlyunder investigation [96]. The pharmacokinetic parametersof 5-AZA-CdR are summarized in Table 5.

5-Aza-CdR in the treatment of hematologic malignancies5-AZA-CdR was first used as a single agent in a phaseI study in children with relapsed or refractory acute

Figure 2 Pharmacokinetics of 5-AZA-CdR at a dose of 15 mg/m2 as a three-hour i.v. infusion to patients with AML and MDS[92]. After i.v. infusion of 5-AZA-CdR, it reaches a steady-stateplasma level within two hours, confirming its very short half-life.5-AZA-CdR, 5-aza-2′-deoxycytidine; AML, acute myeloid leukemia; i.v.,intravenous; MDS, myelodysplastic syndrome.

Table 5 Clinical studies on pharmacokinetics of 5-Aza-CdR

Method(reference)

Dose-schedule Plasma β half-life (minutes)

Clearance(ml/min/kg)

Bioassay[36] 25 to 100 mg/m2 1hour inf q8 hours

35 +/− 5 126 +/− 21

LCMS[92] 15 mg/m2 3hour infq8 hours

35 127-135

HPLC[90] 30 mg/m2 40 to 60hours inf

10 to 15 -

5-AZA-CdR, 5-aza-2-deoxycytidine; inf, infusion; LCMS, liquidchromatography/mass spectrometry.


leukemia [7]. Escalating doses of 5-AZA-CdR between 0.75and 80 mg/kg were explored. When 5-AZA-CdR was givenat doses of 1 to 25 mg/m2/hour for 12 to 30 hours, onlyminor responses were observed. At a dose of 25 to 50 mg/m2/hour for 36 to 44 hours (2.35 to 4.70 μM), twocomplete remissions (CRs) were observed among ninepatients. The authors reported a significant reduction ofcirculating blasts at all dose levels. In addition, 5-AZA-CdRcleared the CSF of two acute lymphocytic leukemia (ALL)patients with central nervous system involvement. No max-imum tolerated dose (MTD) was reported. The major tox-icity was myelosuppression that occurred at doses of 36 to80 mg/kg infused over 36 to 44 hours. In a continuation ofthis study, 27 pediatric acute leukemia patients were treatedwith a continuous infusion of 5-AZA-CdR at doses rangingfrom 37 to 80 mg/kg over 36 to 60 hours. The predictedsteady-state plasma level of 5-AZA-CdR for this schedule is1.5 to 5 μM. The overall objective response rate was 37%(33% in ALL patients and 50% in AML patients) [97]. Inthis phase I trial, the MTD was estimated to be in the rangeof 1,500 to 2,250 mg/m2 [7,97]. Despite promising out-comes, the development of the drug was discontinued dueto the risk of prolonged myelosuppression in patients withadvanced leukemia [98].Several clinical trials investigating different drug dosing

schedules of 5-AZA-CdR demonstrated significant clinicalbenefits in the treatment of patients with MDS and AML[11,56,98-100]. 5-AZA-CdR was initially investigated in atrial of 10 MDS patients [101]. Patients were treated with5-AZA-CdR at a daily dose of 45 mg/m2 divided intothree four-hour infusions for three days (six patients) oras a continuous infusion of 50 mg/m2 for three days (fourpatients). 5-AZA-CdR induced an overall response rate of50% with a complete hematologic response in 40% ofpatients. A subsequent clinical trial reported similar re-sponse rates [102]. The finding that low doses of 5-AZA-CdR (3 mg/kg/cycle) showed activity in patients withsickle cell anemia, by re-expressing the fetal globin gene[10], led to the suggestion that low-dose use of 5-AZA-CdR could be effective against this disease, as well asMDS [18,103]. This hypothesis was evaluated in a phase Istudy with various low-dose levels of 5-AZA-CdR in

patients with MDS or leukemia [11]. Unlike traditionalphase I studies, the goal of this study was not to determinethe MTD, but to determine the optimal biologic dose ofthis drug based on the response and other related para-meters. Forty-eight patients were enrolled, the majoritywith AML. Responses were observed at the intermediatedose level of 15 mg/m2 daily × 10 days (150 mg/m2/course).As a result, this dose level was investigated in 11 additionalpatients. Of the nine responders, eight achieved CR afterone cycle of 5-AZA-CdR. Hematologic toxicities were com-mon but difficult to distinguish from the underlying dis-ease. Non-hematologic side effects were uncommon.Pharmacodynamic studies revealed that dose-dependentdecreases in DNA methylation reached a plateau at ap-proximately 150 to 200 mg/m2/course [104]. Hypomethyla-tion was observed for the p15 tumor-suppressor gene, butthere was no clear correlation between hypomethylation ofthis gene and the overall response [104]. These results sug-gested that response to 5-AZA-CdR can occur at levelsbelow the MTD and that prolonged exposure increases theresponse.5-AZA-CdR was approved by the Food and Drug

Administration (FDA) in 2006 for the treatment ofMDS on the basis of a phase III multicenter trial[105]. In this trial, 170 patients with MDS were rando-mized to receive either i.v. 5-AZA-CdR 15mg/m2 overthree hours every eight hours for three consecutive days(135 mg/m2/course) every six weeks or the best support-ive care (BSC). The overall response rate for the 5-AZA-CdR arm was 30%, compared with 7% for the BSC arm(P <0.001). The median duration of the response was 41weeks with no difference in the time to AML progressioncompared with the BSC [105]. Similarly, the EuropeanOrganization for Research and Treatment of Cancer andthe German MDS Study Group conducted a large phaseIII multicenter trial that randomized 233 elderly patientswith higher-risk MDS to receive either i.v. 5-AZA-CdR15 mg/m2 over three hours every eight hours for threeconsecutive days (135mg/m2/course) every six weeks or theBSC [106]. This trial reported a 34% overall response ratewith 5-AZA-CdR and an improvement in the progression-free survival (0.55 versus 0.25 years; P = 0.004) comparedwith the findings for the BSC group.The time to AML progression and overall survival,

however, did not differ significantly between the twogroups. The main adverse effect due to 5-AZA-CdRtreatment appears to be myelosuppression, including se-vere neutropenia, thrombocytopenia, and anemia. Theincidence of myelosuppression, however, decreases inresponding patients with the continuation of therapy.Grade 3/4 non-hematologic toxicity is rare and usuallyclinically insignificant.MDS patients who fail 5-azacytidine treatment can re-

spond to 5-AZA-CdR. In a study of 14 MDS patients


after the failure of previously administered 5-azacytidinetherapy, low-dose 5-AZA-CdR (20 mg/m2/day i.v. over 5days) was investigated [107]. The overall response ratewas 28% (four patients), including CRs in three patients.In another study, the activity of 5-AZA-CdR was

tested in 27 elderly patients with AML, MDS, or CML[108]. One CR was observed with the dose of 500 mg/m2/24 hours for the three-day schedule (estimatedplasma drug concentration, 2 μM). There were five par-tial remissions. In this mostly ARA-C–resistant patientgroup, the efficacy of 5-AZA-CdR was unquestionable.Myelosuppression, however, was prolonged up to 42days. At the time of this study, G-CSF injections werenot available to accelerate bone marrow recovery.In another study, the activity of 5-AZA-CdR was

tested in 27 elderly patients with AML, MDS, or chronicmyeloid leukemia (CML) [56]. 5-AZA-CdR was adminis-tered at doses of 30 to 90 mg/m2 for AML patients as afour-hour i.v. infusion three times a day for three days.The overall response rate of 45% was encouraging in thisgroup of patients with refractory disease. On the basis ofthese promising outcomes, the same research grouptested 5-AZA-CdR at higher doses in a cohort of 12patients with AML [109]. The overall response rate wasaround 40% in this group of patients with poor progno-ses. The promising results achieved in these two studiessupport the rationale for further investigations of 5-AZA-CdR in AML patients.In a study of elderly AML patients, 5-AZA-CdR was

administered at 20 mg/m2/day for five days every fourweeks [110]. The overall response rate was around 26%.The median time to response was three cycles. Toxicitieswere similar to those in the previous studies at this doselevel. In the study that led to the approval of 5-AZA-CdR for MDS by the FDA, a retrospective detailed inves-tigation of bone marrow revealed that 12 patients withAML were misdiagnosed as MDS. Nine of these patientswere randomly assigned to receive 5-AZA-CdR, and theresponse rate in this subgroup for 5-AZA-CdR was 56%[105]. Several studies have evaluated the combination of5-AZA-CdR with other anti-cancer agents [111]. In thisreview, we have focused primarily on 5-AZA-CdRmonotherapy.Currently, there is no established care for AML

patients who are not eligible to receive standard induc-tion chemotherapy due to poor performance status. Aphase III study that enrolled 485 patients who were atleast 65 years of age with newly diagnosed, de novo, orsecondary AML and intermediate or unfavorable riskcytogenetics was performed [112]. 5-AZA-CdR wasgiven at a dose of 20 mg/m2/day as a one-hour infusionfor five consecutive days every four weeks (estimateddrug plasma concentration of 2 μM for one hour).Patients on 5-AZA-CdR had a median overall survival

(OS) of 7.7 months, compared with 5 months in thecontrol arm with a hazard ratio of 0.85. The stratifiedlog-rank analysis, however, did not demonstrate a statis-tical significance between the groups. Subsequently, anunplanned OS analysis with one year of additionalfollow-up demonstrated the same improvement in me-dian OS with a nominal P value of 0.037 (data on file).As of February 2012, the FDA concluded, based on stat-istical analysis, that 5-AZA-CdR does not appear to im-prove survival in older patients with AML [113]. Thecorrelation of clinical responses and plasma levels of 5-AZA-CdR in hematologic malignancies and in MDS issummarized in Tables 6 and 7, respectively.

5-Aza-CdR in the treatment of solid tumorsThe anticancer activity of 5-AZA-CdR was studied inpatients with refractory and metastatic solid tumors. Ina phase I study of 5-AZA-CdR, the efficacy and toxicityof the drug were also explored in three children withmetastatic solid tumor [7]. 5-AZA-CdR was given bycontinuous infusion. At doses in the range of 0.75 to 80mg/kg for 12 to 44 hours, only limited activity wasobserved.In a study of refractory metastatic prostate cancer

patients, treatment with one-hour infusions of 75 mg/m2 5-AZA-CdR, every eight hours for three doses stabi-lized the disease in 2 of 12 patients with an evaluableresponses [114]. In another study, a seven-day continu-ous infusion of 5-AZA-CdR was investigated in patientswith refractory solid tumors at a daily dose of 2 mg/m2

[115]. The findings of this study demonstrated that gen-omic DNA methylation reverted to baseline levels by 28to 35 days after the start of 5-AZA-CdR treatment. Noobjective responses were observed, however, probablydue to the very low concentrations of 5-AZA-CdR in thebody fluids. Only one patient with metastatic ovariancancer and one patient with renal carcinoma had stabledisease.In a pilot phase I/II study performed in 15 patients

with stage III/IV metastatic non-small cell lung cancer(NSCLC), 5-AZA-CdR was administered over eighthours as a continuous infusion at doses of 200 to 660mg/m2 [116]. The steady-state plasma level of 5-AZA-CdR was estimated to range from 1 to 5 μM. Threepatients survived beyond 15 months, indicating thatrelatively high doses of 5-AZA-CdR had anti-tumor ac-tivity. One patient who received five cycles of 5-AZA-CdR survived for seven years. The hematopoietic toxicityof this schedule was acceptable. These interesting resultswarrant further investigation of 5-AZA-CdR for thetreatment of NSCLC using longer durations of infusions(18 to 24 hours).In summary, treatment of solid tumors with 5-AZA-

CdR led to only limited responses. Notably, however, the

Table 6 Correlation of clinical responses and plasma concentrations of 5-AZA-CdR in patients with hematologicalmalignancies

Type ofleukemia

Dose-schedule Durationinfusion

Estimated plasmaconcentration

Estimated AUC(μM × hour)

Response %ORR(%CR)

References

AMLa ind:135 mg/m2 main:20 mg/m2 1 to 72 hours 0.12 to 1.25 μM 0.12 to 90 26 [143]

AML 20 mg/m2 1 hour 1.25 μM 1.25 25 (24) [110]

AML 20 mg/m2 1 hour 1.25 μM 1.25 64 (47) [144]

CMML 15 mg/m2 3 to 4 hours 0.24 to 0.31 μM 0.72 to 1.24 25(11) [105,145,146]

AML 20 mg/m2 1 hour 1.25 μM 1.25 67 (26) [147]

CML 50 to 100 mg/m2 6 hour 0.52 to 1.0 μM 3.12 to 6.0 B28(10),A55(23),C63(13)

[148]

CML 50 to 100 mg/m2 6 hours 0.52 to 1.0 μM 3.12 to 6.0 53 (0) [121,122]

CML 50 to 100 mg/m2 6 hours 0,52 to 1,0 μM 3.12 to 6.0 84 (10) [121,122]

AMLb 90 mg/m2 4 hours 1.41 μM 5.64 100 (100) [149]

AMLc 125 to 250 mg/m2 6 hours 1.3 to 2.6 μM 7.8 to 15.6 44 (44) [150]

AMLd 125 to 250 mg/m2 6 hours 1.3 to 2.6 μM 7.8 to 15.6 41 (41) [151]

AMLd 125 to 250 mg/m2 6 hours 1.3 to 2.6 μM 7.8 to 15.6 82(73) [137]

AML 250 to 500 mg/m2 6 hours 2.61 to 5.2 μM 15.66 to 31.2 20 (0) [137]

AML 270 to 360 mg/ m2/day t.i.d. 4 hours 4.2 to 5.6 μM 16.8 to 22.4 40 (30) [109]

AML, ALL,CML

300 to 500 mg/m2/day c. i.v. 24 to 120hours

0.16 to 1.3 μM 3.84 to 156.0 26 (4) [108]

AML, ALL 37 to 80 mg/kg c.i.v. 36 to 60hours

1.4 to 5.15 μM 50.4 to 309.0 37 (22) [97]

AML,ALL 0.75 to 80 mg/kg conti.i.v. 12 to 44hours

0.04 to 15 μM 9.0 to 660.0 14 (9) [7]

ain combination with ATRA; bin combination with daunorubicin; cin combination with idarubicin; din combination with amsacrine. 1 μM= 228 ng/ml. 5-AZA-CdR,5-aza-2′-deoxycytidine; A, accelerated; ALL, acute lymphocytic leukemia; AML, acute myeloid leukemia; ATRA, all trans retinoic acid; AUC, area under the curve;B, blastic; C, chronic; CML, chronic myeloid leukemia; CMML, chronic myelomonocytic leukemia; CR, complete remission; ORR, overall response rate.

Table 7 Correlation of clinical responses and plasmaconcentrations of 5-AZA-CdR in patients with MDS

Dose-schedule

Durationofinfusion

Estimatedplasmaconcentration

Response% ORR (%CR)

References

20 mg/m2/dayi.v. q 5d

1hour 1.25 μM 34 (33) [99]

10 mg/m2/dayi.v. q 10d

1hour 0.63 μM

15 mg/m2

i.v t.i.d. x 3d4 hours 0.23 μM 20(15) [123]

15 mg/m2

t.i.d. q3d3 hours 0.31 μM 17 (9) [105]

1 μM = 228 ng/ml. 5-AZA-CdR, 5-aza-2′-deoxycytidine; CR, complete remission;MDS, myelodysplastic syndrome; ORR, overall response rate.


blood concentration of 5-AZA-CdR and the exposuretime to the drug in solid tumor studies were suboptimalwhen compared with the results achieved with higherconcentrations in both in vitro and in vivo animal stud-ies. As clearly demonstrated in the mouse model, dosesof 5-AZA-CdR must be intensified to realize potent anti-tumor activity [60].

Correlation of the plasma level of 5-AZA-CdR withchemotherapeutic actionIn vitro colony-forming assays of leukemic cells revealedthat there is a significant correlation among the concentra-tion of 5-AZA-CdR, the exposure time, and its anticancerand demethylating effects [44,59]. A study on human mye-loid, T-cell and B-cell lines, revealed that 10 μM 5-AZA-CdR for one hour produced a loss of clonogenicity of thesecells lines that was 33%, 50%, and 49%, respectively. Theseobservations confirm the S-phase specificity of 5-AZA-CdR, that is, only cells in S-phase, but not G1 and G2, aretargets of this chemotherapy. During a one-hour treatmentapproximately 50% of the cells are present in S phase. Withan exposure time of 24 hours, 1 μM 5-AZA-CdR produced96%, >98%, and 100% losses of clonogenicity, respectively,for the three leukemic cell lines. These findings indicate

the importance of the exposure time to 5-AZA-CdR,which should be long enough to permit the transit of allleukemic cells into S-phase. The doubling time of the celllines was 20 to 24 hours [117].The therapeutic and toxic effects of 5-AZA-CdR were

investigated in mice with L1210 leukemia. The authors ofone study concluded that a treatment duration of 24hours, giving a 5-AZA-CdR plasma level of around 2 μM,would be most desirable for maximum cell kill [117]. In


another study on L1210 leukemia, the curative dose of5-AZA-CdR was estimated to be 20 mg/kg administeredas an 18-hour infusion [60]. The estimated plasma con-centration of 5-AZA-CdR for this infusion was approxi-mately 3 μM.The antileukemic activity of 5-AZA-CdR was also studied

in the Brown Norway rat leukemia model, which is a goodmodel of human AML [4]. A dose–response relationshipwas observed for 5-AZA-CdR for doses up to 50 mg/kg(administered every 12 hours × 3) in this rat model. Thisdose of 5-AZA-CdR produced a 100% increase in survivaltime. In this rat model of AML, 5-AZA-CdR was more ef-fective than ARA-C (at a dose of 200 mg/kg per treatment).It is interesting that both 5-AZA-CdR and ARA-C reducedthe number of normal hematopoietic stem cells in the bonemarrow by 30% to 40% at a dose of 50 mg/kg, with no fur-ther reduction at higher doses (250 mg/kg). This is a clearindication that the resting normal hematopoietic stem cellssurvive intensive doses of S-phase–specific agents (adminis-tered over an interval of 24 hours) due to the presence ofresting non-proliferating hematopoietic stem cells [4].Studies of the antineoplastic action of 5-AZA-CdR in

mouse models of solid tumors provide some insight intothe importance of the dose schedule. The in vivo/in vitroEMT6 tumor model is an excellent tool to study thepharmacodynamics of antineoplastic agents [118]. In thismodel, EMT6 tumor cells from cell culture are injectedsubcutaneously (s.c.) into mice, and chemotherapy isadministered when the tumor size is approximately 3 to5 mm. After chemotherapy, the tumor is excised andtrypsinized, and single-cell suspensions are plated inPetri dishes. Cell survival after chemotherapy is quanti-fied by comparing the number of colonies to the numberwith controls. Using this assay, 5-AZA-CdR at a dose of30 mg/kg as an 18-hour infusion markedly reduced theproliferative potential of the tumor stem cells [60]. Theestimated plasma level of 5-AZA-CdR in this treatmentwas in the range of 4 μM. These preclinical results illus-trate the importance of using intensive chemotherapywith 5-AZA-CdR to obtain a good antitumor response.

Hematopoietic toxicity of 5-Aza-CdRThe major side effect of 5-AZA-CdR is myelosuppression.In patients with advanced hematologic malignancies, it isdifficult to fully evaluate the hematopoietic toxicity of5-AZA-CdR due to the presence of a large number of ab-normal cells in the bone marrow, which can interfere withrecovery after chemotherapy. Tumor patients without thepresence of malignant cells in the bone marrow are goodcandidates for the evaluation of hematopoietic toxicity on‘normal bone marrow’. Patients with lung cancer withoutprior chemotherapy treated with 660 mg/m2 5-AZA-CdRas an eight-hour infusion exhibited significant recovery ofthe white blood cell count at approximately day 35 [116].

In patients with advanced acute leukemia, the recoveryof the granulocyte count (>500/μl) after an intensivedose of 5-AZA-CdR occurred at about day 45 [97]. Thisobservation suggests that the interval between cycles of5-AZA-CdR therapy should be six weeks to permit ad-equate recovery of the granulocyte count. At low doses of5-AZA-CdR (15 to 20 mg/m2), the drug is well toleratedin patients with MDS, and the primary hematological tox-icity is transient neutropenia, which is predictable andmanageable [92,111,119,120].In a study of patients with CML, 5-AZA-CdR was admi-

nistered at a dose of 500 to 1,000 mg/m2 over five daysand prolonged neutropenia was the major side effect[121,122]. At doses of 75 to 100 mg/m2 delivered over sixhours every 12 hours for 10 doses, the median time toneutrophil recovery above 500/μl was 50 (total dose of1,000 mg/m2/cycle) or 45 days (total dose of 750 mg/m2/cycle), respectively.

Discussion5-AZA-CdR is an effective epigenetic drug for the treat-ment of hematologic malignancies. The clinical efficacy of5-AZA-CdR is due to its demethylating epigenetic action,which reactivates tumor suppressor genes silenced byDNA methylation. Low-dose 5-AZA-CdR (20 mg/m2

one-hour infusion x five days or 15 mg/m2 four-hour infu-sion q8 h × three days) can produce CRs in patients withMDS and AML [99,123]. These low dose schedules causeless toxicity than intensive doses of 5-AZA-CdR. This isespecially important for older patients with a poor per-formance status who are not good candidates for intensivetherapy with 5-AZA-CdR. The interesting responses tolow-dose 5-AZA-CdR suggest that leukemic stem cells arevery sensitive to low concentrations of 5-AZA-CdR, anindication of the immense chemotherapeutic potential ofthis drug.Recent preclinical reports indicate that very low dose

5-AZA-CdR administered frequently (two to threetimes/week) also has the potential to be an effectiveform of therapy for cancer. One reason for the failure ofcytotoxic chemotherapy is that malignant cells can beresistant to the induction of apoptosis due to a non-functional p53 pathway as a result of mutations or dele-tions [124]. However, the genes that program terminaldifferentiation in these apoptosis-resistant malignantcells can be silenced by epigenetic mechanisms, such asDNA methylation, and reactivated by non-toxic doses of5-AZA-CdR. This same treatment maintains the self-renewal of normal hematopoietic stem cells by preventingrepression of stem cell genes by differentiation-inducingstimulus and induces differentiation of AML cells [54].For these reasons the very low dose 5-AZA-CdR does notproduce pronounced granulocytopenia as observed withintense doses of this agent.


Laboratory studies on AML cells support the use of verylow-dose 5-AZA-CdR. 5-AZA-CdR inhibits in vitro prolif-eration, decreases colony formation and induces myeloiddifferentiation of p53-null AML cells [125]. These observa-tions were confirmed using fresh AML cells from a patient.The AML cells were transplanted into NSG immunosup-pressed mice and treated with a s.c. injection of 5-AZA-CdR (0.2 mg/kg three times/week for two weeks, thenonce/week). This very low dose 5-AZA-CdR was muchmore effective in prolongation of the survival time of theleukemic mice than an intense dose of ARA-C (75 mg/kgper day intraperitoneally for five days). The proof ofprinciple of the very low-dose 5-AZA-CdR was also con-firmed in a clinical trial in MDS patients with high-riskcytogenetics [126]. 5-AZA-CdR 3.5 to 7 mg/m2 adminis-tered one to three times/week produced an overall re-sponse of 84% (CR + hematologic improvement + stabledisease), which is remarkable. Complete cytogenetic remis-sions were observed in 50% of the patients. It will be inter-esting to see if this non-toxic differentiation therapy with5-AZA-CdR will be effective in older AML patients whoare not candidates for cytotoxic chemotherapy.One limitation of the low dose 5-AZA-CdR for the

treatment of AML or MDS is the problem of eradicatingmalignant cells in the liver or spleen due to the highactivity of cytidine deaminase. Deamination of 5-AZA-CdR can reduce its concentration to sub-therapeuticlevels in these organs. The use of an inhibitor of cytidinedeaminase, such as tetrahydrouridine, in combinationwith 5-AZA-CdR has the potential to overcome thisproblem [127]. The proof of principle of this approachwas demonstrated in a murine xenotransplant model ofAML where tetrahydrouridine produced a marked en-hancement of the antineoplastic activity of 5-AZA-CdR[128]. The combination of these agents merits a highpriority for clinical investigation in patients withhematologic malignancies.Preclinical studies indicate that very low-dose 5-AZA-

CdR also has the potential to be an effective treatment fortumors with a favorable epigenetic signature. As an ex-ample, human renal carcinoma cells derived from a patientwere inoculated s.c. into nude mice followed by treatmentwith low dose 5-AZA-CdR (0.2 mg/kg s.c. × 3/week) [129].This low dose therapy was very effective in reducing tumorgrowth and did not produce leukopenia. This low dose5-AZA-CdR, in combination with tetrahydrouridine to in-hibit cytidine deaminase, was also very effective in inhibit-ing the growth of murine melanoma tumors in mice [130].The low dose chemotherapy did not produce leukopeniaor reduction in body weight. 5-AZA-CdR was also shownto induce differentiation of both human and murine mel-anoma cell lines.These observations on very low 5-AZA-CdR therapy

of leukemia and tumors were confirmed by Tsai et al.

[131] using a different dose-schedule. These investigatorsshowed that low dose 5-AZA-CdR (72-hour exposure)reduced colony formation of AML cells from patients, butnot the normal hematopoietic stem cells colony-formingunits-granulocyte macrophage (CFU-GM). The low dose5-AZA-CdR (0.1 μM, 72 hours ex vivo) followed by 7 to14 days drug-free media was also shown to decreasetumorigenicity in mouse tumor xenografts.In summary, the very low dose 5-AZA-CdR preclin-

ical studies showed that this type of treatment couldproduce a loss in the self-renewal potential of cancerstem cells due to the increase in the expression of genesthat suppress malignancy. These epigenetic changes aremaintained in the target cells after drug removal andaccumulate with each low dose treatment until there isa complete loss of cancer stem cell potential. The lowdose chemotherapy merits clinical investigation inpatients with cancer. The very low dose 5-AZA-CdRmay also have the potential to maintain CR in patientswith leukemia and arrest malignant progression inpatients with solid tumors. For cancer patients withpoor performance status the very low dose 5-AZA-CdRtherapy may be a good option to improve the quality oflife rather than the use of only supportive therapy or notreatment.Some patients with cancer may not respond or show

disease progression on the low dose-schedule 5-AZA-CdR. This may be due to fact that: a) the cancer may havean epigenetic/genetic signature that is not predisposed tothe induction of terminal differentiation by low dose5-AZA-CdR; b) the target cancer cells may have a lowlevel of deoxycytidine kinase, the enzyme that activatesthe prodrug, 5-AZA-CdR [132]; c) the cancer cells may bein anatomic sanctuaries that have low penetration of5-AZA-CdR (for example, CSF, testis, tumors with a lim-ited blood supply [133]). The concentration of 5-AZA-CdR in these sanctuaries is too low to eliminate the cancerstem cells; d) the cancer cells may be in a biochemicalsanctuary that contains high levels of cytidine deaminase(for example, liver, spleen); e) drug resistance developsmore rapidly after repetitive treatments with low-dosechemotherapy; f ) because 5-AZA-CdR is a cell cycle-specific agent, a one- to four-hour infusion of this agentonly targets cancer cells in S-phase, whereas cells in G1and G2 phases escape the chemotherapeutic action of thisanalog during short-term treatment. A long interval (12 to24 hours) between infusions can also permit leukemicstem cells to pass through the S-phase cell cycle withoutexposure to 5-AZA-CdR and, thus, escape its antileuke-mic action. This possibility was demonstrated in a preclin-ical study on leukemia using the S phase–specific drugARA-C [134].One approach to overcome these caveats is to use in-

tensive chemotherapy with 5-AZA-CdR administered as


a continuous infusion for patients with leukemia. Thisobjective is of high priority and involves determinationof the optimal plasma level of 5-AZA-CdR and durationof treatment that can eliminate leukemic stem cells inthese sanctuaries. The in vitro data on colony assays ofhuman leukemic cell lines indicate that a concentrationof 5-AZA-CdR in the range of 1 to 2 μM for the dur-ation of the cell cycle of the leukemic cells has the abilityto completely eliminate their proliferative potential. An-other approach is to use 5-AZA-CdR in combinationwith an inhibitor of cytidine deaminase, such as tetrahy-drouridine or zebularine, to target the leukemic cells inthe biochemical sanctuaries [135,136]. An additional ap-proach is to use 5-AZA-CdR in combination with a bio-chemical modulator, such as 3-deazauridine, to eliminatedrug-resistant leukemic cells due to a deficiency in de-oxycytidine kinase [132]. Cancer cells deficient in deoxy-cytidine kinase are very sensitive to the cytotoxic actionof 3-deazauridine [132].One of the key points concerning intensive-dose ther-

apy with 5-AZA-CdR is the fact that it produces delayedand prolonged myelosuppression. Several investigatorshave used intensive-dose 5-AZA-CdR in patients withadvanced leukemia and observed that most of thesepatients with a good performance status recovered fromthe hematopoietic toxicity. These early studies were per-formed before the clinical use of granulocyte colonystimulating factors to accelerate the recovery from myelo-suppression. Some examples of the dose schedules ofintensive-dose 5-AZA-CdR that were used are: total doseof 2,479 mg/m2 as a 60-hour i.v. infusion; 500 mg/m2 as a6-hour infusion every 12 hours × 5 days for a total dose of5,000 mg/m2; and a daily dose of 300 to 500 mg/m2 as a24 to 120 hour infusion [97,108,137]. The estimatedplasma level of 5-AZA-CdR in these studies ranged from1 to 3 μM. The hematopoietic toxicity produced by inten-sive doses of 5-Aza-CdR can also be predicted from itscomparative pharmacology with the related deoxycytidineanalogue, ARA-C. Both 5-AZA-CdR and ARA-C areS-phase–specific agents. They have identical metabolism,and their antineoplastic action is due to their incorpor-ation into DNA, but their molecular mechanisms of actiondiffer. 5-AZA-CdR inhibits DNA methylation, whereasARA-C potently inhibits DNA replication. Because theytarget the same cells (proliferating cells in S phase), theyshould produce a similar pattern of hematopoietic toxicity.Most leukemic patients in CR with a good performancestatus recover from the hematopoietic toxicity produced byvery high-dose ARA-C (up to 6,000 mg/m2/day for fourdays; total dose 24,000 mg/m2) [138]. These observationsprovide a rationale for intensive doses of 5-AZA-CdR inthe range of 1,000 mg/m2/day in leukemic patients in CRwithout encountering unacceptable hematologic toxicityfor patients with a good performance status. The recovery

from granulocytopenia after 5-AZA-CdR is approximatelytwo weeks longer than that after ARA-C [97]. This is prob-ably due to the delayed epigenetic action of 5-AZA-CdRon normal hematopoietic stem cells compared with theacute cell kill produced by ARA-C. From this point ofview, it is better to use a six-week interval between cyclesof 5-AZA-CdR rather than the four-week interval used forARA-C.It is a remarkable achievement that current chemother-

apy can induce CR in most patients with hematologic ma-lignancies. The major challenge is maintaining the patientsin CR. Patients in CR are good candidates for experimentalchemotherapy because of their good hematologic status.From an ethical point of view, high-risk leukemic patientswith an unfavorable karyotype that predicts a poor outcomeare good candidates for intensive therapy with 5-AZA-CdR.Pharmacokinetic/pharmacodynamic calculations can

be used to estimate the optimal dose for 5-AZA-CdR.For the initial studies, we recommend a combination ofintensive and low-dose 5-Aza-CdR to treat high-riskpatients with leukemia. For the initial intensive phase,5-AZA-CdR can be infused at a rate of 30 mg/m2/hourfor days one and two (total dose 1,440 mg/m2/day). Thisinfusion rate should give a plasma concentration of ap-proximately 2 μM, shown to be very effective in bothin vitro and in vivo animal studies on leukemia. Theobjective of this intensive therapy of 5-AZA-CdR is totarget the most rapidly proliferating leukemic stem cellsand those in anatomic and biochemical sanctuaries. Theintensive phase is followed by a low-dose phase where5-AZA-CdR is administered on days three and four as ashort infusion at a dose of 30 mg/m2/day. The objectiveof this phase is to target cancer stem cells with a longcell cycle that do not enter the S phase during the firsttwo days of treatment. Supportive care with granulocytecolony stimulating factor is recommended to shortenthe duration of granulocytopenia. The interval betweeneach cycle of therapy should be six weeks to permit ad-equate recovery from bone marrow toxicity. Modifica-tions of the proposed dose schedule may be required foroptimization. In subsequent studies, the low-dose phaseof 5-AZA-CdR can be replaced by histone deacetylase(HDAC) and/or histone methylation inhibitors, whichshowed a synergistic interaction against leukemic cells inpreclinical studies [139,140]. It could also be interestingto design a clinical study in AML patients to see the effi-cacy and safety of decitabine in combination with prom-ising novel tyrosine kinase inhibitors such as quizartinib[141]. Both preclinical and clinical observations indicatethat 5-AZA-CdR has tremendous potential for the treat-ment of hematologic malignancies. The results of thisproposed clinical trial on 5-AZA-CdR will be of greatinterest and will hopefully lead to improved overall sur-vival of patients with advanced leukemia.


Chemotherapy of solid tumors using 5-AZA-CdR alsomerits clinical investigation. Most malignancies have alarge number of tumor suppressor genes that are silencedby aberrant DNA methylation, providing many interestingtargets for 5-AZA-CdR therapy. The preclinical data indi-cate that all types of tumors are sensitive to 5-AZA-CdRtreatment, including low dose therapy. However, the clin-ical responses to low-dose 5AZA-CdR in solid tumors arereported to be very limited. It should be noted, however,that the estimated plasma levels of 5-AZA-CdR in thesetrials was too low. In vitro clonogenic assays using anexposure time of 24 hours on tumor cells indicate thatlow concentrations of 5-AZA-CdR are not very effective.Our preclinical data on chemotherapy of tumors in themouse model suggest that the plasma level of 5-AZA-CdRshould be approximately 3 μM for curative therapy [60].An example of the potential of intensive doses for tumor

therapy is the pilot stage III/IV NSCLC study where thepatients were administered 5-AZA-CdR (660 mg/m2 as aneight-hour infusion), which produced a plasma concentra-tion in the range of 3 μM. This study produced somepromising results: three patients who survived for 15months and one patient who survived for seven years[116]. The preclinical data support the possibility of thistype of response and predict that an infusion time longerthan eight hours should be more effective. One major rea-son for the failure of tumor chemotherapy is the limitedpenetration of drugs into tumors [133]. One approach toovercome this problem is to obtain high plasma concen-trations of anticancer drugs to enhance their penetrationinto tumors. This provides a rationale for the use ofintensive-dose 5-AZA-CdR for the treatment of solidtumors. S phase–specific drugs can be used at very highdoses for a limited duration without unacceptable sideeffects. Patients with metastatic malignancy and poorprognosis are potentially good candidates for intensechemotherapy with 5-AZA-CdR. As an initial study, wesuggest a dose schedule of 60 mg/m2/hour administeredas an 18-hour infusion (total dose of 1,080 mg/m2). Thisdose schedule will give an estimated steady state plasmaconcentration in the range of 4 μM. The interval betweencycles should be six weeks to permit adequate bone mar-row recovery. Patients treated previously with intensivecytotoxic chemotherapy are at risk of severe hematopoietictoxicity and require a minimum of four weeks of recoverybefore being eligible for intensive 5-AZA-CdR therapy.Patients who do not respond to anticancer agents that donot produce hematopoietic toxicity would also be goodcandidates for this investigational therapy. Depending onthe response, the intensive dose schedules for 5-AZA-CdRmay have to be modified for optimization (for example,increase the dose and/or duration of the infusion; use lowdose 5-AZA-CdR between cycles of intensive doses). Thegoal should be to optimize the dose-schedule of 5-AZA-

CdR to reveal its potential for tumor therapy. It may alsobe possible to increase the effectiveness of this tumor ther-apy by using a sequential treatment of 5-AZA-CdR fol-lowed by an inhibitor of histone modification [139,140] orwith a tyrosine kinase inhibitor [142].

ConclusionEpigenetic changes, such as DNA methylation, play a veryimportant role in both leukemogenesis and tumorigenesis.Because these epigenetic changes are very prominent incancer cells and are reversible, they are potential targetsfor chemotherapy with 5-AZA-CdR. Pharmacodynamicand pharmacokinetic analysis of 5-AZA-CdR from bothpreclinical and clinical data can provide insight towardoptimizing cancer treatment using this interesting epigen-etic agent.

Abbreviations5-AZA-CdR: 5-Aza-2′-deoxycytidine; ALL: Acute lymphocytic leukemia;AML: Acute myeloid leukemia; ARA-C: Cytosine arabinoside; BSC: Bestsupportive care; CFU-S: Colony forming unit in spleen; CML: Chronic myeloidleukemia; CMML: Chronic myelomonocytic leukemia; CpG: Cytosine-phosphate-guanine; CR: Complete remission; CSF: Cerebrospinal fluid;D50: 50% decomposition rates; DGU: N′-β-D-2-deoxyribofuranosyl-3-guanylurea; DNMT: DNA methyltransferase; FDA: US Food and DrugAdministration; G-CSF: Granulocyte colony stimulating factor; HDAC: Histonedeacetylase; HPLC: High performance liquid chromatography; HPLC/MS: Highperformance liquid chromatography/mass spectrometry; IC50: Half maximalinhibitory concentration; i.p: Intraperitoneal; MDS: Myelodysplastic syndrome;MTD: Maximally tolerated dose; NFDU: N-(formylamidino)-N′-β-D-2-deoxyribofuranosylurea; NSCLC: Non-small cell lung cancer; OS: Overallsurvival; s.c: Subcutaneous.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsAll authors contributed to the content. All authors read and approved thefinal manuscript.

AcknowledgementsResearch support by a grant from Canadian Cancer Society research grant700795.

Received: 1 October 2012 Accepted: 4 January 2013Published: 1 February 2013

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doi:10.1186/1868-7083-5-3Cite this article as: Karahoca and Momparler: Pharmacokinetic andpharmacodynamic analysis of 5-aza-2’-deoxycytidine (decitabine) in thedesign of its dose-schedule for cancer therapy. Clinical Epigenetics 20135:3.

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